Mapping Proton Wires in Proteins: Carbonic Anhydrase and GFP Chromophore Biosynthesis

Abstract

We have developed an algorithm for mapping proton wires in proteins and applied it to the X-ray structures of human carbonic anhydrase II (CA-II), the green fluorescent protein (GFP), and some of their mutants. For both proteins, we find more extensive proton wires than typically reported. In CA-II the active site wire exits to the protein surface, and leads to Glu69 and Asp72, located on an electronegative patch on the rim of the active site cavity. One possible interpretation of this observation is that positively charged, protonated buffer molecules dock in that area, from which a proton is delivered to the active site when the enzyme works in the dehydration direction. In GFP we find a new internal proton wire, in addition to the previously reported wire involved in excited state proton transfer. The new wire is located on the other face of the chromophore, and we conjecture that it plays a role in chromophore biosynthesis that occurs following protein folding. In the last step of this process, transient carbanion formation was suggested to occur on the bridge carbon [Pouwels et al. Biochemistry 2008, 47, 10111]. Residues on the new wire (Thr62, His181, Arg96) may participate in proton abstraction from this bridge carbon atom. A possible mechanism involves a rotation of the Thr62 side chain and completion of a short wire by which the proton is transported to His181, while the negative charge is transferred to the imidazolone carbonyl, producing a homoenolate intermediate that is stabilized by Arg96. Finally, comparison of the proton wires in the two proteins reveals common motifs, such as short internalized Ser/Thr-Glu hydrogen-bonded pairs for ultrafast proton abstraction, and threonine side chain rotation functioning as a proton wire switch.

Proton-Coupled Electron Transfer in Organic Synthesis: Fundamentals, Applications, and Opportunities

“…These experiments elucidate not only the role for hydrogen bonding in proton transfer, but also corroborate work by Thomas demonstrating increased stability of hydrogen-bound phenoxyl radicals [129]. Proton movement in PCET processes is limited to short (i.e., hydrogenbonding contact) distances [130]; however, hydrogen-bonding networks are frequently used as "proton wires" [131] to accomplish long-range proton transfer and are well documented in GFP [132], ferredoxin I of Azotobacter vinelandii [133], cytochrome c oxidase [134], and numerous other proteins [135]. In a notable example, a hydrogen-bonding network between Tyr122 and Cys439 in ribonucleotide reductase (RNR) allows for a net proton transfer over > 30 Å [136].…”

Reductive activation of the heme iron–nitrosyl intermediate in the reaction mechanism of cytochrome c nitrite reductase: a theoretical study

“…Keeping in mind that the calcium site is directly connected to the active-site Tyr 218 and that the distance to the catalytic iron center is only 10.7 Å , the calcium site may also play an important role in the proton supply. Moreover, a similar proton transfer role has been previously established and clearly discussed for the zinc-containing active site of carbonic anhydrase [21].…”

A Proton Wire and Water Channel Revealed in the Crystal Structure of Isatin Hydrolase

“…The manganese-binding site was formed by His-73, His-77, and Asp-79. A strong peak (17) in the anomalous difference density map at this position (Fig. 3a) confirmed the presence of manganese over calcium in the binding site, although calcium is present in large excess in the crystallization conditions.…”

“…A number of proton wires have been identified and studied in other systems, e.g. gramicidin A (16), carbonic anhydrase II, and green fluorescent protein (17). To facilitate the proton transfer in biological systems, proton wires were formed.…”